Further investigations of the effect of pressure on retention in ultra-high-pressure liquid chromatography

In this study, we investigated further the large increases in retention with pressure that we observed previously in RP-LC especially for ionised solutes. These findings were initially confirmed on a conventional silica C-18 Column, which gave extremely similar results to the hybrid C-18 phase originally used. Large increases in retention factor of similar to 50% for a pressure increase of 500 bar were also shown for high MW polar but neutral solutes. However, experiments with the same bases in ionised and non-ionised forms Suggest that somewhat greater pressure-induced retention increases are found for ionised solutes. Retention increases with pressure were found to be considerably smaller for a C-1 column compared with a C-18 Column: decreases in retention with increasing pressure were noted for ionised bases when using a bare silica column in the hydrophilic interaction chromatography (HILIC) mode. These observations are consistent with the partial loss of the solvation layer in RP-LC as the solute is forced into the hydrophobic environment of the stationary phase, and consequent reduction in the solute molar volume, while the water layer on the surface of a HILIC packing increases the hydration of a basic analyte. Finally, retention changes with pressure in RP-LC can also be observed at a mobile phase pH close to the solute pK(a), due to changes in pK(a) with pressure. However, this effect has no influence on the results Of most Of our studies. (C) 2009 Elsevier B.V. All rights reserved.